A spectroscopic study of divalent ion binding sites in synaptic membranes is proposed. The sensitized fluorescence of terbium will be used to monitor specific calcium and other divalent ion binding sites. Analysis of fluorescence data in the presence of physiological ions will provide dissociation constants for those ions. The release of cations from these sites by opiates will be investigated in vivo after peripheral administration of agonists and in vitro with synaptosomes and synaptic membranes. The experiments have been designed to distinguish between direct and indirect displacement of cations. Measurement of the degree of hydration of bound Tb3 ion by fluorescence quenching experiments in D2O-H2O mixtures will give information about the localization of the sites on the basis of their hydrophobicity. Further characterization of the cation binding sites will include an estimation of possible clustering of the sites and the involvement of specific amino acids.